The structural behavior of cytochrome c is studied to better understand protein folding in general, as well as to the probe the specific roles of cytochrome c in cellular respiration and apoptosis. Mutation of the phenylalanine at position 82 in yeast cytochrome c perturbs the heme environment by increasing the exposure of the heme to solution and lowering its reduction potential. If a histidine is introduced at this position, the resulting mutant exhibits redox-dependent ligand switching. The reduced form prefers a near-native ligation of Met80 at the iron, but oxidation promotes the replacement of Met80 by His82. This His82-ligated form has a substantially lower potential than the Met80-ligated form. We are able to monitor the ligand exchange process in solution using cyclic voltammetry and functionalized gold electrodes. Oxidation of the reduced Met80-ligated form is followed by conversion to the His82-ligated form. Reduction of the oxidized His82-ligated form is followed by very rapid conversion to the Met80-ligated form. By varying the potential scan rate we can access different time domains for oxidation and reduction. At modest scan rates the two different forms each contribute an irreversible signal to the voltammetry, separated by about 350 mV. However, at high scan rates, we can outrun the ligand exchange and capture nearly reversible oxidation and reduction for the His82-ligated form. We have used digital simulation of the voltammetry to deduce ligand exchange rates for this system. In addition, we have examined the pH dependence of this system. Lowering the pH from 6 to 4 appears to greatly destabilize the structure of this mutant, lowering the reduction potential obtained for the Met80-bound form. This acid-induced destabilization occurs at a higher pH range than for wild-type cytochrome c.

Redox-induced ligand switching in F82H cytochrome <i>c</i>

247th National Meeting (2014)

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Redox-induced ligand switching in F82H cytochrome c